MS CouplingEnhances ChiralMethod Development

Smaller particles and the addition of MS detection help speed chiral analysis time and accelerate
method development.

» by Waters Corp., Milford, Mass.

Chirality plays a critical role in drug efficacy because enantiomers may have vastly different biological, pharmacological and toxico- logical properties. The majority of newly introduced drugs are chi- ral, and, by 2020, according to a report by Global Industry Analysts,
Inc., it is expected that nearly 95% of pharmaceutical drugs will be chiral.
To keep pace with this growth, researchers in drug discovery are seeking
faster methods to enable higher throughput enantioselective analyses, enantiomeric excess determination and impurity profiling of chiral molecules.

Separation of enantiomers (compounds with the same molecular weight
that are mirror images of each other) poses a difficult chromatographic
challenge. Chiral separation method development is typically less predictable than achiral method development. Small differences in molecular
structure can significantly change resolution for a given column/mobile
phase system. Furthermore, analysts often encounter many structurally
diverse stereoisomers with purity levels varying from 70 to 90 percent.

Chiral method development is traditionally accomplished by tediously
testing various column chemistry and mobile phase combinations, using
normal-phase liquid chromatography (NPLC) to find a system that will
separate the enantiomers of interest. Then, the mobile phase and gradient composition is fine-tuned to optimize the method. Supercritical-fluid
chromatography (SFC), a form of NPLC, has shown superior resolving
power in chiral separations and obviates the use of toxic solvents, such as
hexane and chloroform, typically associated with NPLC. SFC mobile phases
are primarily compressed carbon dioxide to which may be added small
amounts of a co-solvent, such as methanol. However, the inability to meter
supercritical CO2 and control key parameters such as temperature, pressure,
flow rate and gradient composition—reliably and reproducibly—
complicates and slows SFC method development. This has hindered the adoption
of SFC as a routine analytical tool.

Waters’ UltraPerformance Convergence Chromatography (UPC2) system raises the performance bar for NPLC separations using dense gas CO2
as the mobile phase. It has the ability—especially by using small-particle
columns and coupling the high specificity of mass spectrometry with UV
detection—to shorten separation cycle time, thereby facilitating and speeding up chiral separation methods development. The UPC2 system combines
the best features of LC and GC: higher selectivity via orthogonal modes of
separation and higher mobile phase diffusion and efficiency, respectively.

When adequately compressed and made dense, CO2 becomes a green,non-toxic, inexpensive alternative to HPLC-grade solvents, especially thosetoxic organic liquids that incur significant disposal costs. As the primaryUPC2 mobile phase, its low viscosity may decrease operating pressure whileincreasing efficiency for a given particle size and linear velocity. Comparedto LC solvents, mass transfer in supercritical-fluid CO2 is enhanced. Itscritical temperature (31 C) is far lower than typical GC operating tempera-tures, so, as with LC, heat-labile compounds may be analyzed. Perhaps themost significant advantage of CO2 may be economy. It reduces the cost ofthe mobile phase used per sample analysis from dollars to pennies.

Figure 1: UPC2 analysis of flurbiprofen enantiomers. Reducing the particle
size from 5 to 3 µm and the column internal diameter from 4.6 to 2.1 mm,
while maintaining the same flow rate (2 mL/min) reduces analysis time from
about 3.5 min to 34 sec.